Congenital anomalies associated with the lower urinary tract, such as posterior urethral valves (PUV), neuropathic bladders (i.e. myelomeningocoele) and bladder exstrophy, have long-term effects on bladder function, despite advances in surgical treatment and patient management.  Preliminary work with biopsy samples from exstrophy bladders undergoing secondary repair showed extensive tissue fibrosis and its replacement of smooth muscle.  However, there is no evidence of contractile failure of the remaining smooth muscle [1].  The underlying reason for the failure of bladder function associated with other congenital anomalies remains unknown.  We hypothesised that increased fibrosis is a significant cause of bladder contractile failure for a range of congenital anomalies.  This was tested with in vitro experiments on detrusor from children with and without congenital anomalies by comparing the magnitude of contractile responses and histological measurement of smooth muscle and connective tissue content.

Bladder biopsy samples were from six patient cohorts: i) normal bladder function (ureteric implantation, excision of urachal cysts: n=14; 8 male, 6 female; median age 33 months); ii) PUV (n=12; all male; median age 61 months); iii) neuropathic bladder (n=10; 7 male, 3 female; median age 76 months); iv) secondary procedures for exstrophy repair (n=18; 12 male, 6 female; median age 81 months); v) primary exstrophy repair (n=5; 4 male, 1 female; 1-3 days); vi) cloacal anomaly (n=4; all female; median age 65 months).  Ages were statistically similar for all cohorts, except those for primary exstrophy repair. Biopsy samples were cut from the lateral bladder wall, the mucosa removed and placed in Ca-free Tyrode’s solution. 

A strip was immediately dissected (≈1 mm diam; 5 mm length) for functional experiments and any remaining tissue placed in 10% formaldehyde solution for histology.  The muscle strip was superfused with Tyrode's solution (including 24 mM NaHCO3/5% CO2, pH 7.4, 36°C) and isometric contractions recorded.  Nerve-mediated contractions (abolished by 1 µM tetrodotoxin) were generated  by electrical field stimulation (EFS) with 0.1 ms pulses delivered in 3-s trains at 1-24 Hz.  Contractures to the cholinergic receptor agonist carbachol (0.1-30 µM) or the purinergic (P2X1) receptor agonist alpha, beta, ß-methylene ATP (ABMA, 10 µM) were generated in unstimulated preparations. Contraction magnitudes were normalised to cross-section area and quoted as mN.mm-2. Fixed samples were dehydrated, mounted in paraffin blocks: 5 µm sections were mounted on TESPA-coated glass slides and stained with Elastin van Gieson (collagen, red; elastin, black; muscle yellow/orange).  Smooth muscle and connective tissue (collagen/elastin) areas were measured with colour filters in the Image-J program.  Three separate regions (50x50 µm) were analysed and the average used.

Data are mean ± SEM (n=number of biopsies).  Multiple data sets were analysed by ANOVA with Dunnett post-hoc tests to compare against data from normal bladders.  A Spearman rank-order correlation coefficient, r, was calculated to assess association between variables.  Force-frequency or concentration-response curves were fitted to: T=(Tmax.x^m)/(x^m+k^m); Tmax is the maximum response at high stimulation frequency (f) or agonist concentration (S); x is the different values of f or S; k is the value of x (frequency or carbachol concentration) required to achieve Tmax/2; m is a constant (^ represents raised to the power of m).  Curve-fitting used an iterative program (KaleidaGraph) to estimate Tmax and k values.  k-values for carbachol are equivalent to EC50 values and are expressed as pEC50 (=-logEC50).

Maximum contractions (Tmax) to EFS (figure 1A) or carbachol stimulation (figure 1B) were reduced in preparations from all congenital anomalies, except for cloacal anomaly, when compared to data from functionally normal bladders.  The frequency-dependence of EFS contractions was similar in all preparations, as assessed by similar values of k.  The concentration-dependence to carbachol was also similar in all preparations, as assessed by pEC50 values, except for samples from neuropathic bladders where carbachol was more potent (pEC50 values greater; figure 1C).  Contractile responses to 10 µM ABMA showed a similar profile to those with carbachol, i.e. smaller in preparations from all congenital anomaly patients, except for those with cloacal anomaly.  Atropine-resistant EFS contractions, at 16 Hz stimulation, were recorded in samples from all cohorts and the percentages of the contraction in the absence of atropine were all similar (figure 1D). The smooth muscle:connective tissue (SM:CT) ratios were greatest in detrusor from normal bladder function (2.29±0.37) and cloacal anomaly (2.12±0.40) cohorts, reduced to statistically similar values in the PUV (0.86±0.17), neuropathic (1.20±0.20) and secondary exstrophy (0.56±0.07) cohorts and to even smaller values for the neonatal exstrophy (0.18±0.04) cohort.  A plot of EFS Tmax values as a function of SM:CT ratio (fig 2) showed a high degree of positive association between the SM:CT ratio and contraction magnitude (r=0.95, p<0.05, n=7), or a coefficient of determination (r^2) of 88%.

Reduced contractile function is a feature of detrusor smooth muscle obtained from children with a range of congenital disorders, except for cloacal anomaly.  Reduced function was observed whether contractions to agonists (carbachol or ABMA) or excitation of embedded nerves was used.  It is therefore unlikely that a particular pathway in contractile activation was attenuated as the normalised reduction of force was similar in all modalities.  However, there was a significant association between contraction magnitude and proportion of smooth muscle in the biopsy samples.  The value for the coefficient of determination between contraction magnitude and SM:CT ratio was 88%.  This may be interpreted as replacement of smooth muscle with connective tissue accounts for the great majority of the loss of contractile function.  Such an importance of this single factor to account for contractile dysfunction in this group of patients suggests that an understanding of why increased deposition of connective tissue occurs and how it may be avoided or reversed is of paramount importance.

The most important determinant of reduced contractile function in detrusor samples from children with congenital anomalies is loss of muscle tissue and replacement by connective tissue.  Therapeutic strategies to reduce connective tissue content should be an effective way to improve bladder function in these patients.

Johal NS, Arthurs C, Cuckow P, Cao K, Wood DN, Ahmed A, Fry CH.  J Pediatr Urol 2019; 15: 154.e1-154.e9.